Higher order coiling or supercoiling exists in the double-stranded DNA of all organisms as well as some viruses. Negative supercoiling imparts a torsional strain which can produce open or single-stranded regions at specific sites in DNA. These regions may serve as recognition sites for regulatory proteins and enzymes involved in DNA replication, transcription and recombination and may have other functions. The long-term goal of this proposal is to examine the presence, nature and biological significance of single-stranded regions in supercoiled DNA. To achieve this goal, we will (1) evaluate three different single-strand specific endonucleases as probes for open regions in pBR322 DNA by determining the nucleotide sequences cleaved and the effects of environmental conditions and superhelical density on the site specificity, (2) probe the structure of other supercoiled DNAs to test the generality of our findings, and (3) enzymatically delete single-stranded regions to directly test their biological importance in vivo. Knowledge of the relationships between DNA supercoiling, DNA structure and biological function is essential for understanding the regulation and execution of DNA functions in normal and cancer cells.